Thermostable repair enzyme for oxidative DNA damage from extremely thermophilic bacterium, Thermus thermophilus HB8.
The mutM (fpg) gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 266 amino acid protein with a molecular mass of approximately 30 kDa. Its predicted amino acid sequence showed 42% identity with the Escherichia coli protein. The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.coli one, whereas the number of Pro residues, considered to increase protein stability, was increased. The T.thermophilus mutM gene complemented the mutability of the E.coli mutM mutY double mutant, suggesting that T. thermophilus MutM protein was active in E.coli. The T.thermophilus MutM protein was overproduced in E.coli and then purified to homogeneity. Size-exclusion chromatography indicated that T. thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution. Circular dichroism measurements indicated that the alpha-helical content of the protein was approximately 30%. Thermus thermophilus MutM protein was stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C. Denaturation analysis of T.thermophilus MutM protein in the presence of urea suggested that the protein had at least two domains, with estimated stabilities of 8.6 and 16.2 kcal/mol-1, respectively. Thermus thermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.
Mikawa
T
T
Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560, Japan.
Kato
R
R
Sugahara
M
M
Kuramitsu
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D029968:Escherichia coli Proteins; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli